GeneAmp PCR Carry-over Prevention Kit from Applied Biosystems

The GeneAmp PCR Carry-over Prevention Kit provides reagents for a simple yet powerful method of ensuring that PCR products cannot be reamplified in subsequent PCR amplifications, thereby preventing false positive results. This kit uses an enzymatic reaction analogous to the restriction-modification and excision-repair systems of cells to specifically degrade PCR products from previous PCR amplifications in which dUTP has been incorporated, without degrading native nucleic acid templates.

The method used to render PCR products susceptible to degradation involves substituting dUTP for dTTP in the PCR mixture, and pretreating all subsequent PCR mixtures with the enzyme, AmpErase Uracil N-glycosylase (UNG) prior to PCR amplification.1 Products from PCR amplification contain uracil, and are readily distinguishable from native thymidine-containing DNA templates. Products from previous PCR amplifications are eliminated by excising uracil residues using UNG, and degrading the resulting abasic polynucleotide with heat. Although UNG is active on single- and double-stranded dU-containing DNA,2 ribouracil residues in RNA and dUTP itself are not substrates for UNG.3

AmpliTaq and AmpliTaq Gold DNA polymerase, and the other components of the PCR reaction mixture remain intact during UNG treatment, providing PCR amplification free of PCR product carry-over. The dU-containing PCR product behaves like dT-containing DNA in blotting, cloning, sequencing, and most other post-PCR analyses.4

1. Longo, M.C., et al. 1990. Gene 93:125-128
2. Varshney, U., et al. 1988. J. Biol. Chem.263:7,776-7,784.
3. Lindahl, T., et al. 1977. J. Biol. Chem.252:3,286-3,294.
4. Erlich, H.A. et al. 1991. Science 252: 1,643-1,651. For Research Use Only. Not for use in diagnostic procedures.